ASSAY VALIDATION - Biomarker Assay Validations – A Time for Change?

INTRODUCTION

Biomarkers have been used
for many years in drug development and delivery for a wide range of clinical
utilities, and throughout the past decade, their use has substantially
increased. To accurately assess the measurement performance and characteristics
and determine the range of conditions under which the biomarker will provide
reproducible and accurate data, analytical methods for biomarkers must be
validated. In 1991, the Food and Drug Administration (FDA) made new guidance
available for bioanalytical method validation, which informed all laboratories doing
bioanalysis how they should validate their scientific methods, its central
focus being on methods for the evaluation of Pharmacokinetics (PK) – [Guidance
updated 2001 & 2013 (draft)]. Since that time, it has been the “holy grail”
for almost all researchers working in this scientific arena. However, it has
also been used by many laboratories in the same way for validating biomarker
assays for drug discovery and development. Recently, there has been considerable
debate within the community over whether the guidance is applicable or best
scientific practice to areas outside of PK evaluation. The following explores
these debates and consider whether it is time to re-evaluate the requirements
for biomarker assay validation.

WHAT IS A BIOMARKER?

The World Health Organization
(WHO) defines a biomarker as, “a characteristic that is objectively measured
and evaluated as an indicator of normal biological processes, pathogenic processes,
or pharmacological responses to a therapeutic intervention.”1 In practice,
biomarkers include tools and technologies that ultimately help to build an understanding
of the prediction, cause, diagnosis, and progression of disease and the outcome
of treatment.2

Molecular biomarkers can take
many forms and have become a pivotal tool in basic and clinical research as
well as in clinical practice. In today’s research environment, the use of
biomarkers for many different clinical utilities in clinical trials has become widely
accepted, and they are fast becoming an essential part of clinical development.

THE DRUG DISCOVERY & DEVELOPMENT LANDSCAPE

Almost 10 years ago, the
pharmaceutical industry was facing a remarkably high attrition rate for drugs
in clinical development. Multiple studies were reporting that clinical success
rates across the drug industry could potentially be even lower than estimated previously.3
Despite major advances in the basic science of drug discovery and development,
which led to a substantial increase in the number of new drug targets, the
development of novel effective therapies did not appear to be following the
same upward trajectory. In 2006, it was estimated that only 8% of tested
products entering Phase I trials gained regulatory approval, and many of these
failures happened in late-stage clinical trials.4 Additionally, very
few drugs were making it out of the clinical research pipeline, and in 2007, the
US FDA approved only 17 new molecular entities and two biologic licenses; the
lowest number since 1983.5

The significant reduction
in clinical success rates across the drug industry appeared to be caused by a
gap in the industry’s ability to predict a drug candidate’s early performance.
To counteract the downturn in novel effective therapies, it was suggested that
biomarkers, typically used to monitor therapeutic progress, disease
progression, and the efficacy of interventions, could provide a solution.
Biomarkers were considered attractive as they may predict drug efficacy more
quickly than conventional clinical endpoints, and have the potential to
substantially accelerate production development in certain disease areas.
Furthermore, by identifying candidates that are likely to fail earlier in the process,
biomarkers can lead to a reduction in drug development costs. As the mantra goes
with drug development: if you fail early, you fail cheap.6

Biomarker assay
requirements are designed before clinical trials commence, and a new drug would
not be developed without simultaneously looking for biomarkers for efficacy,
safety, and to measure the pharmacodynamics (PD) of the drug. Other utilities
are also used (depending upon the mode of action of the drug for instance). The
field of oncology is leading the way in the use of biomarkers in drug development,
and their use as an alternative to clinical endpoints in drug development has
meant that oncology has not experienced the same downturn in drug development
that has been experienced by other therapeutic areas. However, many of the
biomarker determinations in this arena are not performed using “wet” methods
for quantitative assays in biological fluids.

THE BIOMARKER VALIDATION CHALLENGE

Validation is the process
of assessing the biomarker analytical method and its measurement performance
characteristics, as well as determining the range of conditions under which the
biomarker will give reproducible and accurate data.7 The validation of
biomarker analytical methods is a crucial step in the quest to deliver high-quality
research data, and the criteria for validation are defined by the following:

-The nature of the
question that the biomarker is designed to address

-The degree of certainty
that is required for the desired answer

-The assumptions about the
relationship between changes in the biomarker and clinical endpoints or other
clinical utilities (eg, mechanistic, PD, etc)8

Although it is perfectly
clear that method validation is a crucial step when using biomarkers, there is
an absence of official guidelines for the validation of biomarker assays. Since
the FDA 2013 Draft guidance was published, its content in relation to biomarker
assays has been the subject of wide and contentious debate within the industry.
This ultimately has culminated in inconsistent adaptations of related regulations
in bioanalytical and clinical laboratories. It has been agreed for many years that
there is a lack of standardization between laboratories. This was the original motivation
behind the FDA releasing the FDA Guidance for Industry for Bioanalytical Method
Validation [originally 1991, updated 2001 and 2013 (draft)], as mentioned.
Essentially, these documents have driven forward improvements in the
standardization of bioanalytical methods, and researchers continue to use the
FDA (and other regulators) guidance(s) today.

However, although the
guidance has undoubtedly proven beneficial, it only addresses critically the
validation of assays to support PK assessments. Despite the document implying
its limited scope (above) for purposes other than PK evaluation, many laboratories
still continue to use the guidance verbatim, as it seems to have been interpreted
that it is the only way that analytical methods should be validated. As the use
of biomarkers for drug development accelerated and some researchers continued
to use the FDA guidance, many clinical scientists were simultaneously
questioning the extensive and confusing application of the terms “biomarker”
and “validation,” and whether this guidance was appropriate for biomarker
analytical methods used in drug discovery and development.

ADDRESSING THE CHALLENGES

Successful validation
requires an understanding of exactly what an analytical method is doing and how
it works. Once this is known, experiments can be designed to test the method
and prove the performance of the assay. A further step that needs to be
considered when utilizing biomarkers is the clinical validity of the results.
Validation should demonstrate that a method is “reliable for the intended
application.” In 2000-2001, a group of scientists working under the auspices of
the American Association of Pharmaceutical Scientists (AAPS) recognized that by
following the FDA guidance for industry for the validation of Biomarker
methods, we were often not able to answer the clinical questions being asked,
nor ensure the delivery of the clinical utility of the biomarker being studied.

These scientists brought
together by a Bioanalytical Focus Group of the AAPS wanted to publish a
document to bring consensus as to how biomarker assays should be validated to
ensure results obtained were clinically relevant. The outcome was a whitepaper
titled Fit-for-Purpose Method Development
and Validation for Successful Biomarker Measurement.9 This was
the first seminal document that had been published on this subject (specifically
for drug development) that highlighted a number of issues that have also been
recognized in other white papers published since then: the “potential need to
step out of the framework of regulated bioanalysis guidelines” as it was
important to: “keep in mind the intended use of the data and the attendant
regulatory requirements associated with that use.”

Ultimately, the whitepaper
advised scientists how biomarker methods should be developed and validated when
used in drug development as opposed to using them in diagnostics, to ensure
that they were ‘fit-for-purpose.’ Many laboratories referred to this document
and since Lee and colleagues published their whitepaper, the Global CRO Council
(GCC) and European Bioanalysis Forum (EBF) also published papers on biomarker
assay validation.10,11 Overall, there has been a drive forward in
the number of laboratories demonstrating ways in which they generate improved
data, thanks to this documentation being available (GCC survey 2016 –
unpublished data).

TIME FOR A CHANGE

Despite an increase in
awareness about biomarker assay validation, there are still multiple instances
in which laboratories are obtaining incorrect, inaccurate, or variable results
because they are following PK guidance documents for assay validation, which
potentially can present a serious concern. The author has seen multiple
examples in which biomarker results data has been generated that was
incompatible with life, or inaccurate data has been produced that would lead to
a false interpretation of the results from a clinical perspective. This is not
good (nor acceptable) to the company developing the drug or the subjects
participating in the clinical trials. This of course not only demonstrates the
shortfalls of using a PK assay validation document for biomarker methods, but
also that some bioanalytical laboratories do not have scientists with clinical
knowledge that is crucial in the biomarker field. This is one of the reasons
why understanding physiology and clinical biochemistry is so important to
ensure that reliable and appropriate biomarker results data are generated using
methods validated to appropriate standards.

By way of an example of
one of the major issues from a clinical and scientific standpoint in using the
PK guidance is that no weight is given to the different physiological changes
seen in different biomarkers, nor the different performance characteristics of
different methods when we set acceptance criteria for QC samples used when
analyzing patient samples. The degree of change seen in different biomarkers is
often specific to each biomarker from a clinical significance perspective –
they are not all the same. For example, of ~400 biomarkers that are well
characterized and understood, intra-individual variability in normal subjects
range from <1% to >90%. Using PK guidance, however, the acceptance criteria
for all the biomarkers using the same technology would be the same (eg,
LC-MS/MS +/- 15% to 20%, and Immunoassay +/- 20% to 25%). Not only does this
not make clinical sense, but it doesn’t add up statistically either because different
analytical methods – even within the same technology – perform differently, and
yet, the known analytical performance (from the validation experiments) is not
being taken into account when setting acceptance criteria for QCs in sample batches.
If acceptance criteria was primarily based on known method performance, this
would prove methods are working as they should be but also give confidence limits
around the results being reported, aiding statistical and clinical
interpretation. In summary on this point, the concept of statistically valid
Quality Control does not exist if we follow the guidance, and clinical relevance
is not covered in any way in determining if the method is fit for its intended purpose.

Discussions surrounding
the topic of biomarker assays and validation continue to grow, and throughout
the past 12 or more months, the industry has begun to speak out on the subject
of biomarker assay validation in a unified way. In September 2015, the FDA and
AAPS organized Crystal City VI, called in response to the previous meeting
(Crystal City V).12 At Crystal City V, a revised version of FDA guidance
published in 2013 was discussed. The document was a source of concern and
disagreement from those involved in biomarker science, due to the content and
also the recommendations within the paper with regard to biomarker assay validation
guidance. At Crystal City VI, some very central points were raised.13
However, the major point that was raised by several key opinion leaders was
that biomarker assays differ from PK assays, meaning that they should be
validated in a different way. It was agreed that when using biomarkers for drug
development and delivery, several important points should be considered (Table
1).

Crystal City VI appeared
to be the first time there had been a major shift in momentum. It appeared that
scientists were willing to speak out in a unified voice, and a large majority
were giving the same message; it was time for a change to the current guidance.
Crystal City VI was followed by a number of additional meetings (WRIB April
2016, AAPS-NBC May 2016, and EBF BM workshop June 2016), featuring discussions
that expanded on the points raised, and questioned how we go about moving
forward. Industry key opinion leaders (KOLs) are now at a point where they have
identified and are in consensus that there are numerous issues that need to be
addressed, and in order to do so, it would be beneficial for a different
guidance document to be considered. The significant question within the industry
now is how do we move forward?

The limited scope within
the existing guidance for purposes other than PK evaluation and increasing use
of biomarkers for drug discovery and development means we are now at a time at
which, in my opinion, a change in industry guidance for biomarker assay
validation is essential.

THE FUTURE OF BIOMARKER VALIDATION

Despite the increased use
of biomarkers, it appears that many researchers are still continuing to use the
FDA guidance document for validation even though it only critically addresses
the validation of assays to support PK evaluation and also has a limited scope
described within the document in terms of studies where it should be used.

There have been a number
of clinical studies in which the data obtained has been unrepresentative and
incorrect because the bioanalytical lab has followed PK guidance to validate
their bioanalytical methods. This is extremely concerning and highlights the
importance of ensuring that laboratories conducting research have a real
investment in terms of the right team, who understand the clinical questions
being asked, and have the know-how to develop and validate methods that will
answer the necessary questions.

As researchers, our aim is
to follow the principles of good science, and to ensure that the results
obtained are clinically robust and relevant. The use of biomarkers for drug
discovery and development is a hot topic within the industry, and the FDA
guidance has been questioned on many occasions in relation to its use for the
validation of Biomarker assays. Now is the time to listen to the questions that
have been raised and work toward an updated recommendation from industry KOLs
who hopefully regulators will consider in developing revised guidance documents
that will improve the reliability of biomarker results and ultimately benefit
overall advances in healthcare in assisting the development of new drugs.

REFERENCES

1. WHO International
Programme on Chemical Safety Biomarkers in Risk Assessment: Validity and
Validation. 2001. http://www.inchem.org/documents/ehc/ehc/ehc222.htm.

John L.
Allinson is the Head of Biomarker Strategy of LGC and brings over 40 years
of experience in developing and working with biomarkers, including their use in
drug development across all therapeutic categories and phases of development.
Before joining LGC, Mr. Allinson spent 22 years in NHS Clinical Pathology
services before moving into the CRO industry, where he managed Central,
Bioanalytical, and Biomarker Laboratories at a number of CROs. He was part of
the AAPS Ligand Binding Assay Bioanalytical Focus Group (LBABFG) Biomarker Committee,
which published the first consensus white paper on biomarker assay validation
requirements in Drug Development, and was also co-author of the recently
published first white paper on the Validation of Multiplexed Biomarker assays.
He has been an invited presenter at over 50 international conferences and continues
to present an educational workshop on Biomarker Assay Method Development and
Validation at the World Biomarker Congress.